Gene identification using the yeast two-hybrid system

Methods in Enzymology

Volumn 273, Issue , 1996, Pages 331-347

  Bai, Chang ^a   Elledge, Stephen J ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

BETA GALACTOSIDASE;

ARTICLE; DNA BINDING; GENETIC ANALYSIS; HYBRID; NONHUMAN; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN PROTEIN INTERACTION; TRANSCRIPTION INITIATION; YEAST;

EID: 0029842096     PISSN: 00766879     EISSN: None     Source Type: Book Series    
DOI: 10.1016/s0076-6879(96)73029-x     Document Type: Article

References (23)

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13. J. T. Mulligan and S. J. Elledge, in "Molecular Genetics of Yeast: A Practical Approach," p. 65. Oxford University Press, Oxford, 1994.
14. PEG can be briefly heated in a microwave oven to help it go into solution if necessary.
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16. We observe that nitrocellulose filter becomes more brittle in liquid nitrogen than do nytran filters. However, nitrocellulose filters adhere to colonies slightly better than does nytran. Filters can be thoroughly washed with deionized water and reused.
17. It is best to have at least medium-size colonies with which to perform this assay.
18. R. D. Gietz and R. A. Woods, in "Molecular Genetics of Yeast: A Practical Approach." Oxford University Press, Oxford, 1994.
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20. SC-Trp medium is used to select for pAS-X but YPED gives the best transformation efficiencies. The doubling time for Y190 (pAS-X) in YPED varies between 2.0 to 3.5 hr depending on the plasmid it carries.
21. We have found that total yeast RNA works more reproducibly as a carrier but more work is required to prepare than DNA. Protocols for preparing ssDNA and yeast RNA for transformation can be found in Refs. 15 and 16, respectively.
22. Cells in PEG are more fragile and often die when pelleted; thus the recovery step is useful. Plating directly from the PEG without the recovery step also works, but is more messy. Cells lose less than half their viability when frozen and can be stored indefinitely. This is useful because they can be thawed and plated at a optimal density for screening/selection at leisure.
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